PROJECT SUMMARY/ABSTRACT BPA is ubiquitously found in many consumer products. Humans are exposed to BPA daily and more than 90% of surveyed individuals have detectable levels of BPA in their urine. Obesity, metabolic disorders, and neurological disorders have been associated with BPA exposure in humans and animals. In rodents, exposure to BPA of F0 pregnant females results in obesity phenotypes in the F2 generation that persist in the unexposed F3 and subsequent generations. These observations suggest that exposure to BPA induces epigenetic alterations in the F1 germline that can be inherited inter- and transgenerationally. However, the mechanisms by which these epigenetic alterations are established, maintained, and transmitted to subsequent generations remain largely unexplored. To address this question, we exposed pregnant mouse females to BPA during embryonic days 7-13, when the germline of the fetus is being reprogrammed. We find that exposure to BPA leads to an obese phenotype that is transmitted maternally and paternally for at least 4 generations. Sperm from the obese progeny of BPA-exposed mice contain new chromatin accessible sites not present in unexposed controls corresponding to new binding sites for CTCF and several nuclear hormone receptors. Importantly, these new sites are present in sperm of F2-F5 mice, suggesting that they are directly or indirectly responsible for the obesity phenotype. Together, these results suggest that epigenetic alterations induced by BPA can be transmitted transgenerationally by the male germline. However, we do not know how these alterations are initially acquired in the germline during reprograming or how they are maintained in the absence of the original stimulus by BPA. Here we propose experiments to study how BPA-induced epigenetic changes are established and maintained in early germ line cells. The work proposed in Aim 1 will identify new transcription factor (TF) binding sites induced by BPA exposure at different time points during the development of the Primordial Germ Cells (PGCs), prospermatogonia, and Spermatogonial Stem Cell (SSCs), and examine whether the changes in TF binding are associated with changes in the transcriptome of these cells. In Aim 2, I will study the relationship between changes in TF binding in PGCs and alterations of DNA methylation, histone modifications, and histone variants at new TF sites throughout the differentiation of the male germline. Aim 3 will analyze the maintenance of epigenetic alterations in the germline of the F2 generation in the absence of BPA exposure, and the temporal relationship between TF binding and changes in DNA methylation and chromatin structure. The knowledge gained from the proposed studies will reveal for the first time how BPA- induced epigenetic alterations are established during epigenetic reprogramming in the embryonic germline and maintained in the male germ cells of subsequent generations. These studies will be highly significant in understanding the mechanisms of transgenerational inheritance of epiphenotypes induced by endocrine disrupting chemicals (EDCs), a first step towards designing interventions to reverse these effects.